Multiple E3 ligases act as antiviral factors against SARS-CoV-2 via inducing the ubiquitination and degradation of ORF9b.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) open reading frame 9b (ORF9b) antagonizes the antiviral type I and III interferon (IFN) responses and is ubiquitinated and degraded via the ubiquitin-proteasome pathway. However, E3 ubiquitin ligases that mediate the polyubiquitination and degradation of ORF9b remain unknown. In this study, we identified 14 E3 ligases that specifically bind to SARS-CoV-2 ORF9b. Specifically, three E3 ligases, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, induced K48-linked polyubiquitination and degradation of ORF9b, thereby attenuating ORF9b-mediated inhibition of the IFN response and SARS-CoV-2 replication. Moreover, each E3 ligase performed this function independent of the other two E3 ligases. Therefore, the three E3 ligases identified in this study as anti-SARS-CoV-2 host factors provide novel molecular insight into the virus-host interaction.IMPORTANCEUbiquitination is an important post-translational modification that regulates multiple biological processes, including viral replication. Identification of E3 ubiquitin ligases that target viral proteins for degradation can provide novel targets for antagonizing viral infections. Here, we identified multiple E3 ligases, including HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, that ubiquitinated and induced the degradation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 9b (ORF9b), an interferon (IFN) antagonist, thereby enhancing IFN production and attenuating SARS-CoV-2 replication. Our study provides new possibilities for drug development targeting the interaction between E3 ligases and ORF9b.

The Experience of Cervical Cancer Patients Undergoing Hysterectomy: A Qualitative Study.

INTRODUCTION: Due to different social and cultural backgrounds, cervical cancer patients' experience of the treatment process and quality of life after treatment will be different. This study sought to gain in-depth understanding of the experiences of Chinese cervical cancer patients as regards their quality of life and physical symptoms. METHODOLOGY: Semi-structured interviews were used to collect data. We recruited 15 women with cervical cancer in eastern China for in-depth interviews. All data were entered into the NVivo 12 software program for analysis. RESULTS: Four themes emerged from the data: (a) uncertainty; (b) physical suffering; (c) psychological pressure; and (d) challenges of marriage and family. DISCUSSION: Cervical cancer patients showed concerns about the disease itself and the physical discomfort it causes, as well as changes in social relations. Health professionals need to talk about these issues and develop strategies to address them accordingly.

CRL4B E3 ligase recruited by PRPF19 inhibits SARS-CoV-2 infection by targeting ORF6 for ubiquitin-dependent degradation.

The accessory protein ORF6 of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key interferon (IFN) antagonist that strongly suppresses the production of primary IFN as well as the expression of IFN-stimulated genes. However, how host cells respond to ORF6 remains largely unknown. Our research of ORF6-binding proteins by pulldown revealed that E3 ligase components such as Cullin 4B (CUL4B), DDB1, and RBX1 are potential ORF6-interacting proteins. Further study found that the substrate recognition receptor PRPF19 interacts with CUL4B, DDB1, and RBX1 to form a CRL4B-based E3 ligase, which catalyzes ORF6 ubiquitination and subsequent degradation. Overexpression of PRPF19 promotes ORF6 degradation, releasing ORF6-mediated IFN inhibition, which inhibits SARS-CoV-2 replication. Moreover, we found that activation of CUL4B by the neddylation inducer etoposide alleviates lung lesions in a SARS-CoV-2 mouse infection model. Therefore, targeting ORF6 for degradation may be an effective therapeutic strategy against SARS-CoV-2 infection.IMPORTANCEThe cellular biological function of the ubiquitin-proteasome pathway as an important modulator for the regulation of many fundamental cellular processes has been greatly appreciated. The critical role of the ubiquitin-proteasome pathway in viral pathogenesis has become increasingly apparent. It is a powerful tool that host cells use to defend against viral infection. Some cellular proteins can function as restriction factors to limit viral infection by ubiquitin-dependent degradation. In this research, we identificated of CUL4B-DDB1-PRPF19 E3 Ubiquitin Ligase Complex can mediate proteasomal degradation of ORF6, leading to inhibition of viral replication. Moreover, the CUL4B activator etoposide alleviates disease development in a mouse infection model, suggesting that this agent or its derivatives may be used to treat infections caused by SARS-CoV-2. We believe that these results will be extremely useful for the scientific and clinic communities in their search for cues and preventive measures to combat the COVID-19 pandemic.

Predictive role of NICU-related stress, postpartum depression trajectory and family coping on growth trajectory of moderate-to-late preterm infants: A longitudinal study.

AIMS: To describe the changes in moderate-to-late preterm infants' (MLPIs) growth during 12 months of corrected age (CA) and to examine the predictive role of NICU-related stress, postpartum depression trajectory and family coping ability on the physical developmental trajectory of MLPIs. DESIGN: A prospective longitudinal study. METHODS: There were 237 mother-infant dyads with at least two follow-up data records included. General characteristics and NICU-related stress were recorded from medical records at baseline. Infants' physical growth was measured at 40 weeks, 1, 3, 6, 9 and 12 months CA during outpatient follow-up. Maternal postpartum depressive symptoms and family coping ability were assessed by questionnaires at 1, 3, 6, 9 and 12 months CA and 1 month CA respectively. We investigated the modifiable factors inside and outside of NICU on the trajectories of physical growth in the first year in MLPIs, mainly by using latent growth curve models with time-varying covariates. RESULTS: The curved trajectories of weight, length and head circumference in the first year in MLPIs demonstrated gradually slowed growth rates and these infants were above the WHO growth standards for the same age and sex. The latent growth curve models indicated that more NICU-related stress was negatively associated with the weight and length at 40 weeks CA, and family coping ability (parent-child relationship) at 1 month CA was associated with the growth rate of weight. Besides, more NICU-related stress predicted faster length growth rate. The infants of mothers who were in the group of high-level postpartum depression trajectory had a slower growth rate of head circumference. CONCLUSIONS: Our study identified the modifiable factors along the care continuum influencing the trajectory of MLPIs' physical growth. Nurses should receive more training about infant stress measurement and family-centred care to work in partnership with parents so that MLPIs can reach their full developmental potential. Also, multidisciplinary interventions including stress reduction strategies, close psychological monitoring and education improving parent-infant relationships should be further developed to achieve optimizing growth in the first year of MLPIs. IMPLICATIONS FOR THE PROFESSION AND/OR PATIENT CARE: It is recommended that nurses pay attention to the long-term physical growth status of MLPIs, and closely support their families. Quantifying NICU-related stress and developing reduction strategies should be the priority for clinical staff during hospitalization. After discharge, persistent screening of depressive symptoms, psychological intervention and education about the parent-child relationship need to be included in the follow-up visits. PATIENT OR PUBLIC CONTRIBUTION: No patient or public contribution. The study only included patients who were research participants.

Deubiquitinase USP1 regulates sarbecovirus ORF6 protein function.

SARS-CoV-2 belongs to the subgenus Sarbecovirus, which universally encodes the accessory protein ORF6. SARS-CoV-2 ORF6 is an antagonist of the interferon (IFN)-mediated antiviral response and plays an important role in viral infections. However, the mechanism by which the host counteracts the function of ORF6 to restrict viral replication remains unclear. In this study, we found that most ORF6 proteins encoded by sarbecoviruses could be ubiquitinated and subsequently degraded via the proteasome pathway. Through extensive screening, we identified that the deubiquitinase USP1, which effectively and broadly deubiquitinates sarbecovirus ORF6 proteins, stabilizes ORF6 proteins, resulting in enhanced viral replication. Therefore, ubiquitination and deubiquitination of ORF6 are important for antagonizing IFN-mediated antiviral signaling and influencing the virulence of SARS-CoV-2. These findings highlight an essential molecular mechanism and may provide a novel target for therapeutic interventions against viral infections.IMPORTANCEThe ORF6 proteins encoded by sarbecoviruses are essential for effective viral replication and infection and are important targets for developing effective intervention strategies. In this study, we confirmed that sarbecovirus ORF6 proteins are important antagonists of the host immune response and identified the regulatory mechanisms of ubiquitination and deubiquitination of most sarbecovirus ORF6 proteins. Moreover, we revealed that DUB USP1 prevents the proteasomal degradation of all ORF6 proteins, thereby promoting the virulence of SARS-CoV-2. Thus, impeding ORF6 function is helpful for attenuating the virulence of sarbecoviruses. Therefore, our findings provide a deeper understanding of the molecular mechanisms underlying sarbecovirus infections and offer potential new therapeutic targets for the prevention and treatment of these infections.

Deubiquitinase USP39 promotes SARS-CoV-2 replication by deubiquitinating and stabilizing the envelope protein.

The SARS-CoV-2 envelope (E) protein is highly conserved among different viral variants and important for viral assembly and production. Our recent study found that the E protein is ubiquitinated and degraded by the E3 ligase RNF5 through the proteasome pathway. However, whether E ubiquitination can be reversed by host deubiquitinase has not yet been determined. Here, we identify by mass spectrum analysis that the deubiquitinases USP14 and USP39 specifically interact with E, while USP39 potently reverses E polyubiquitination. USP39 interacts with E via the arginine-rich motif (AR) and deubiquitinates E polyubiquitination via the inactive ubiquitin-specific protease domain. Therefore, USP39 protects E from RNF5-mediated degradation, resulting in the enhancement of E stability and E-induced cytokine storms. Moreover, loss-and-gain assays demonstrated that USP39 promotes the replication of various SARS-CoV-2 strains by stabilizing protein level of E that can be ubiquitinated but not other viral proteins. Our findings provide useful targets for the development of novel anti-SARS-CoV-2 strategies.

Hypermethylated CDO1 and CELF4 in cytological specimens as triage strategy biomarkers in endometrial malignant lesions.

Objective: Developing a non-invasive and reliable triage test for endometrial malignant lesions is an important goal, as it could help to reduce the number of invasive diagnostic procedures required and improve patient survival. We aimed to estimate the diagnostic value of DNA methylation levels in cervical cytological samples of endometrial cancer (EC) and endometrial atypical hyperplasia (AH). Methods: A total of 607 women who had indications for endometrial biopsy in the Department of Obstetrics and Gynecology of Cangzhou Central Hospital from October 2022 to April 2023 were enrolled in this study. The cervical exfoliated cells were collected for gene methylation before endometrial biopsy. Clinical information, tumor biomarkers, and endometrial thickness (ET) of transvaginal ultrasonography (TVS) were also collected. With endometrial histopathology as the gold standard, multivariate unconditional logistic regression was applied to analyze the risk factors of endometrial malignant lesions. The role of cysteine dioxygenase type 1 (CDO1) and CUGBP Elav-like family member 4 (CELF4) gene methylation as a triage strategy biomarker in endometrial malignant lesions was specifically explored. Results: Multivariate logistic regression analysis showed that premenopausal ET ≥ 11 mm or postmenopausal ET ≥ 5 mm, CDO1 ΔCt ≤ 8.4, or CELF4 ΔCt ≤ 8.8 were the risk factors for AH and EC, with odds ratios (ORs) (95%CI) of 5.03 (1.83-13.82) and 6.92 (1.10-43.44), respectively (p-values < 0.05). The sensitivity and specificity of CDO1/CELF4 dual-gene methylation assay for AH and EC reached 84.9% (95%CI: 75.3%-94.5%) and 86.6% (95%CI: 83.8%-89.5%), respectively. ET combined with DNA methylation detection further improved the specificity to (94.9%, 95%CI: 93.1%-96.8%). Conclusion: The accuracy of cervical cytology DNA methylation is superior to that of other clinical indicators in the non-invasive examination of endometrial malignant lesions. DNA methylation combined with TVS can further improve the specificity and is a promising biomarker triage strategy in women with suspected endometrial lesions.

Prevalence of psychological problems among caregivers of children and adolescents with neurodevelopmental disorders during the COVID-19 pandemic: A meta-analysis and systematic review.

BACKGROUND: The COVID-19 pandemic impacted children and adolescents with neurodevelopmental disorders, which caused difficulties and increased caregivers' burden. AIM: The objective of this study was to assess the prevalence of psychological problems among caregivers of children and adolescents with neurodevelopmental disorders. Methods and procedures We searched the PubMed, Embase and Web of Science databases for relevant studies published from December 2019 to March 2023. Random effects models were used to calculate the pooled prevalence of psychological problems among caregivers. Subgroup analyses were used to detect potential heterogeneity and sensitivity analyses were performed to evaluate the robustness of the included studies. Egger's and Begg's tests were used to examine publication bias. Outcomes and results Twenty studies involving 14,743 participants were included in this systematic review and meta-analysis. The main psychological problems among caregivers were anxiety (36.6%, 95% confidence interval [CI] 19.6-53.7%), depression (41.1%, 95%CI 35.4-46.8%), and stress (58.9%, 95%CI 45.1-72.7%). There were differences in prevalence by study year, national economic level, continent, and sample size. Conclusions and implications Our study showed that caregivers of children and adolescents with neurodevelopmental disorders had a high prevalence of anxiety, depression, and stress during the COVID-19 pandemic. Therefore, the psychological problems of these caregivers should not be overlooked. We recommend that the government should provide caregivers with more medical and financial assistance. What this paper adds? The current study is the first systematic review and meta-analysis focusing on parents whose children have neurodevelopmental disorders during the COVID-19 pandemic. The results show that the prevalence of psychological problems among caregivers of children with neurodevelopmental disorders is particularly prevalent, which suggests that we should attach importance to the parenting pressure and mental health of this special group.

Ring Finger Protein 146-mediated Long-chain Fatty-acid-Coenzyme a Ligase 4 Ubiquitination Regulates Ferroptosis-induced Neuronal Damage in Ischemic Stroke.

Ischemic stroke (IS) is one of the leading causes of disability and death worldwide. Long-chain fatty-acid-coenzyme A ligase 4 (ACSL4) is a critical isozyme for ferroptosis that participates in the progression of IS. RING finger protein 146 (RNF146) is an E3 ligase predicted to interact with ACSL4 and regulated by activating transcription factor 3 (ATF3). The molecular mechanism of the RNF146/ACSL4 axis in IS is still unclear. Oxygen-glucose deprivation/reperfusion (OGD/R) treatment was used as the in vitro model, and middle cerebral artery occlusion (MCAO) mice were established for the in vivo model for IS. The protein level of ACSL4 was monitored by Western blot during ischemic injury. RNF146 was overexpressed in vitro and in vivo. The interaction of RNF146 and ACSL4 was determined by co-immunoprecipitation (Co-IP) assay. Chromatin immunoprecipitation (ChIP) assay and luciferase assay were utilized to determine the regulation of ATF3 on RNF146. Ferroptosis was evaluated by the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), Fe2+, and protein levels of related genes including ACSL4, SLC7A11, and GPX4. ACSL4 was downregulated upon OGD treatment and then increased by re-oxygenation. RNF146 was responsible for the ubiquitination and degradation of ACSL4 protein. RNF146 overexpression could prevent the stimulation of OGD/R-induced LDH, MDA, and Fe2+ levels and ferroptosis-related gene expression. ATF3 could activate the transcription and expression of RNF146, leading to the inhibition of OGD/R-induced neuron ferroptosis. The ATF3-mediated RNF146 could alleviate neuronal damage in IS by regulating ACSL4 ubiquitination and ferroptosis, providing a novel theoretical basis for exploring therapeutic targets and strategies.

Astragaloside IV alleviates neuronal ferroptosis in ischemic stroke by regulating fat mass and obesity-associated-N6-methyladenosine-acyl-CoA synthetase long-chain family member 4 axis.

Ischemic stroke (IS) is a detrimental neurological disease with limited treatment options. Astragaloside IV (As-IV) was a promising bioactive constituent in the treatment of IS. However, the functional mechanism remains unclear. Here, IS cell and mouse models were established by oxygen glucose deprivation/re-oxygenation (OGD/R) and middle cerebral artery occlusion (MCAO). Quantitative reverse transcription PCR (RT-qPCR), Western blotting, or Immunofluorescence staining measured related gene and protein expression of cells or mice brain tissues, and the results revealed altered expression of acyl-CoA synthetase long-chain family member 4 (Acsl4), fat mass and obesity-associated (Fto), and activation transcription factor 3 (Atf3) after treatment with As-IV. Then, increased N6 -methyladenosine (m6 A) levels caused OGD/R or MCAO were reduced by As-IV according to the data from methylated RNA immunoprecipitation (MeRIP)-qPCR and dot blot assays. Moreover, through a series of functional experiments such as observing mitochondrial changes under transmission electron microscopy (TEM), evaluating cell viability by cell counting kit-8 (CCK-8), analyzing infract area of brain tissues by 2,3,5-triphenyltetrazolium chloride (TTC) staining, measuring levels of malondialdehyde (MDA), lactate dehydrogenase (LDH), Fe2+ , solute carrier family 7 member 11 (Slc7a11) and glutathione peroxidase 4 (Gpx4) and concentration of glutathione (GSH), we found that Fto knockdown, Acsl4 overexpression or Atf3 knockdown promoted the viability of OGD/R cells, inhibited cell ferroptosis, reduced infract size, while As-IV treatment or Fto overexpression reversed these changes. In mechanism, the interplays of YTH N6 -methyladenosine RNA-binding protein 3 (Ythdf3)/Acsl4 and Atf3/Fto were analyzed by RNA-pull down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assay. Fto regulated the m6 A levels of Acsl4. Ythdf3 bound to Acsl4, and modulated its levels through m6 A modification. Atf3 bound to Fto and positively regulated its levels. Overall, As-IV promoted the transcription of Fto by upregulating Atf3, resulting in decreased m6 A levels of Acsl4, thus, improving neuronal injury in IS by inhibiting ferroptosis.

The trajectories of physical growth in 4 months postnatal corrected age among preterm infants discharged from neonatal intensive care units and associated factors: A prospective study.

Objectives: Growth retardation is a risk for premature infants. In addition to demographic and perinatal factors, preterm infants' physical growth may be affected by neonatal intensive care unit (NICU) stress, maternal postpartum depression, and mother-infant interaction. This study aimed to investigate the trajectories of physical growth in 4 months corrected age among preterm infants discharged from the NICU and the impactors on these trajectories. Methods: A prospective study was conducted among 318 preterm infants from September 2019 to April 2021 in Shanghai, China. Latent growth modeling was applied to identify the weight, length, and head circumference growth trajectories in 4 months corrected age and explore the effects of demographic and medical characteristics, infant stress during NICU stay, maternal postpartum depression, and mother-infant interaction on each trajectory. Results: Unconditional latent growth models showed curve trajectories with increasingly slower growth in weight, length, and head circumference until 4 months of corrected age. Conditional latent growth models showed that a longer length of stay in the NICU and more skin punctures were negatively associated with weight at 40 weeks corrected gestational age (ß = -0.43 and -0.19, respectively, P < 0.05). The maternal postpartum depression between 40 weeks corrected gestational age and 1 month corrected postnatal age was associated with a lower growth rate of length (ß = -0.17, P = 0.040), while between 2 and 3 months corrected postnatal age, there were lower growth rates of weight and head circumference (ß = -0.15 and -0.19, respectively, P < 0.05). The mother-infant interaction scores between 40 weeks corrected gestational age and 1 month corrected postnatal age negatively predicted the growth rate of weight (ß = -0.19, P = 0.020). Conclusion: The physical growth trajectories of preterm infants discharged from the NICU were influenced by infant stress during the NICU stay, maternal postpartum depression and mother-infant interaction.

The E3 ligase RNF5 restricts SARS-CoV-2 replication by targeting its envelope protein for degradation.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a severe global health crisis; its structural protein envelope (E) is critical for viral entry, budding, production, and induction of pathology which makes it a potential target for therapeutics against COVID-19. Here, we find that the E3 ligase RNF5 interacts with and catalyzes ubiquitination of E on the 63rd lysine, leading to its degradation by the ubiquitin-proteasome system (UPS). Importantly, RNF5-induced degradation of E inhibits SARS-CoV-2 replication and the RNF5 pharmacological activator Analog-1 alleviates disease development in a mouse infection model. We also found that RNF5 is distinctively expressed in different age groups and in patients displaying different disease severity, which may be exploited as a prognostic marker for COVID-19. Furthermore, RNF5 recognized the E protein from various SARS-CoV-2 strains and SARS-CoV, suggesting that targeting RNF5 is a broad-spectrum antiviral strategy. Our findings provide novel insights into the role of UPS in antagonizing SARS-CoV-2 replication, which opens new avenues for therapeutic intervention to combat the COVID-19 pandemic.

Characterization and analysis of linear epitopes corresponding to SARS-CoV-2 outbreak in Jilin Province, China.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants have caused hundreds of thousands of deaths and shown serious social influence worldwide. Jilin Province, China, experienced the first wave of the outbreak from December 2020 to February 2021. Here, we analyzed the genomic characteristics of the SARS-CoV-2 outbreak in Jilin province using a phylogeographic tree and found that clinical isolates belonged to the B.1 lineage, which was considered to be the ancestral lineage. Several dominant SARS-CoV-2 specific linear B cell epitopes that reacted with the convalescent sera were also analysed and identified using a peptide microarray composed of S, M, and E proteins. Moreover, the serum of convalescent patients infected with SARS-CoV-2 showed neutralizing activity against four widely spreading SARS-CoV-2 variants; however, significant differences were observed in neutralizing activities against different SARS-CoV-2 variants. These data provide important information on genomic characteristics, linear epitopes, and neutralizing activity of SARS-CoV-2 outbreak in Jilin Province, China, which may aid in understanding disease patterns and regional aspects of the pandemic.

Deubiquitinase ubiquitin-specific protease 3 (USP3) inhibits HIV-1 replication via promoting APOBEC3G (A3G) expression in both enzyme activity-dependent and -independent manners.

BACKGROUND: Ubiquitination plays an essential role in many biological processes, including viral infection, and can be reversed by deubiquitinating enzymes (DUBs). Although some studies discovered that DUBs inhibit or enhance viral infection by various mechanisms, there is lack of information on the role of DUBs in virus regulation, which needs to be further investigated. METHODS: Immunoblotting, real-time polymerase chain reaction, in vivo / in vitro deubiquitination, protein immunoprecipitation, immunofluorescence, and co-localization biological techniques were employed to examine the effect of ubiquitin-specific protease 3 (USP3) on APOBEC3G (A3G) stability and human immunodeficiency virus (HIV) replication. To analyse the relationship between USP3 and HIV disease progression, we recruited 20 HIV-infected patients to detect the levels of USP3 and A3G in peripheral blood and analysed their correlation with CD4 + T-cell counts. Correlation was estimated by Pearson correlation coefficients (for parametric data). RESULTS: The results demonstrated that USP3 specifically inhibits HIV-1 replication in an A3G-dependent manner. Further investigation found that USP3 stabilized 90% to 95% of A3G expression by deubiquitinating Vif-mediated polyubiquitination and blocking its degradation in an enzyme-dependent manner. It also enhances the A3G messenger RNA (mRNA) level by binding to A3G mRNA and stabilizing it in an enzyme-independent manner. Moreover, USP3 expression was positively correlated with A3G expression ( r  = 0.5110) and CD4 + T-cell counts ( r  = 0.5083) in HIV-1-infected patients. CONCLUSIONS: USP3 restricts HIV-1 viral infections by increasing the expression of the antiviral factor A3G. Therefore, USP3 may be an important target for drug development and serve as a novel therapeutic strategy against viral infections.

Development and Internal Validation of a Model Predicting the Risk of Recurrent Stroke for Middle-Aged and Elderly Patients: A Retrospective Cohort Study.

OBJECTIVE: To develop and validate a model for predicting the risk of recurrent stroke among middle-aged and elderly stroke patients. METHODS: A total of 1,327 stroke patients from the China Health and Retirement Longitudinal Study (CHARLS) were included in the retrospective cohort study, and they were randomly divided into the training and test sets at a ratio of 7:3. Univariate and multivariate regression analyses were used to select the predictors in the training set, which were used to develop logistic regression model. The Delong test and area under the receiver operating characteristic curve were adopted to investigate the predicted performance of the model. RESULTS: The average follow-up time was 2.26 ± 0.52 years, and the incidence of recurrent stroke was 14.47%. The result indicated that duration of moderate exercise, duration of walking, social activities, and diastolic blood pressure were associated with the risk of recurrent stroke among the middle-aged and elderly stroke patients. A logistic regression model was constructed to predict the risk of recurrent stroke after 2 years: [Logit (PR)=ln (PR/(1-PR) =-1.658-0.841 moderate exercise (<2 hours/day)-0.559∗moderate exercise (≥2 hours/day)-0.906∗walk (<2 hours/day)-1.131∗walk (≥2 hours/day)-0.474∗social activities 1-0.968∗social activities 2-1.248∗social activities 3 + 0.015∗diastolic blood pressure)]. The value of the area under the curve reached 0.75, showing that the logistic regression model performs well in the prediction of the risk of recurrent stroke. CONCLUSIONS: A logistic regression model for predicting the risk of recurrent stroke was developed among middle-aged and elderly stroke patients after 2 years, and the model showed good discrimination and accuracy via internal validation.

Study on the mechanism of Ginseng-Gegen for mesenteric lymphadenitis based on network pharmacology.

Background: This study aimed to determine the main active ingredients of the Ginseng-Gegen (Panax Ginseng-Radix Puerariae) drug pair, to predict relevant action targets, and to establish a network of "drug-active ingredients-targets", to ultimately explore the mechanism of Ginseng-Gegen in the treatment of mesenteric lymphadenitis. Methods: The Traditional Chinese Medicine Systems Pharmacology (TCMSP) platform was used to screen the chemical constituents of Ginseng-Gegen, and the active ingredient targets were retrieved by UniProt database. The databases of GeneCards and the Online Mendelian Inheritance in Man (OMIM) were applied to search for mesenteric lymphadenitis-related targets. Cytoscape software was used to construct the network of active ingredient-action targets. The biological functions of the targets were analyzed in the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database. Results: A total of 26 potential active ingredients of the Ginseng-Gegen drug pair were screened, with 128 drug-related targets and 255 mesenteric lymphadenitis-related targets. After matching, 23 potential targets were obtained for treating mesenteric lymphadenitis. Among them, MOL012297 (puerarin), MOL005344 (ginsenoside Rh2), and MOL000358 (beta-sitosterol) were linked to 3 or more key target genes. They were supposed to be important ingredients of Ginseng-Gegen in the treatment of mesenteric lymphadenitis. Conclusions: Ginseng-Gegen is related to oxidative stress and inflammation, and it is a part of the nuclear factor κB (NF-κB) signaling pathway, tumor necrosis factor (TNF) signaling pathway, and the advanced glycation end products/receptor for advanced glycation end products (AGE-RAGE) signaling pathway. These biological processes and signaling pathways may be potential mechanisms of Ginseng-Gegen for treating mesenteric lymphadenitis.

Sex-Specific Differences in the Clinical Profile Among Patients with Tracheobronchial Tuberculosis: A Hospital-Based Cross-Sectional Study in Shenzhen, China.

Purpose: Tracheobronchial tuberculosis (TBTB) has been proposed to occur more commonly in female patients. However, to date, studies that systematically delineate differences between female and male patients with TB infection are lacking. We aimed to comprehensively assess the sex-specific differences in clinical manifestation, bronchoscopy performance, bacteriological examination, and imaging of TBTB in Shenzhen, China. Methods: All patients with diagnosed TBTB from August 1, 2018 to July 31, 2021 at The Third People's Hospital of Shenzhen were enrolled in the present study. Demographic information, clinical manifestations, blood tests, chest computed tomography, and bronchoscopic findings were collected, and assessed their sex-specific differences. Results: Of these 331 patients, 238 patients (71.9%) were female, and 93 patients (28.1%) were male, with an overall average age of 37.3 years. The average age of male patients with TBTB was more than 5 years older than that of female patients. The prevalence of lymph fistula and diabetes mellitus was significantly higher in male patients than female patients (8.6% vs 1.7%, P = 0.005; 17.2% vs 2.1%, P < 0.001). The positive proportion of sputum smear was higher in male patients (27.9%) than in female patients (16.7%, P = 0.026). Moreover, the mean monocyte-to-lymphocyte ratio, serum CRP, and IL-6 levels were significantly higher in male patients than in female patients (P < 0.05). Conclusion: In summary, in patients with TBTB diagnosis, male sex was associated with a high prevalence of diabetes mellitus, lymph fistula, and smear-positive ratio, as well as high inflammation levels. The management of young female and male patients with diabetes mellitus and high inflammation levels should be strengthened. Furthermore, to reduce the burden of TBTB, we must pay attention to the risk of TBTB in past tuberculosis patients, especially male patients under 45 years old and female patients over 45 years old.

The Deubiquitinase USP29 Promotes SARS-CoV-2 Virulence by Preventing Proteasome Degradation of ORF9b.

Ubiquitin signaling is essential for immunity to restrict pathogen proliferation. Due to its enormous impact on human health and the global economy, intensive efforts have been invested in studying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its interactions with hosts. However, the role of the ubiquitin network in pathogenicity has not yet been explored. Here, we found that ORF9b of SARS-CoV-2 is ubiquitinated on Lys-4 and Lys-40 by unknown E3 ubiquitin ligases and is degraded by the ubiquitin proteasomal system. Importantly, we identified USP29 as a host factor that prevents ORF9b ubiquitination and subsequent degradation. USP29 interacts with the carboxyl end of ORF9b and removes ubiquitin chains from the protein, thereby inhibiting type I interferon (IFN) induction and NF-κB activation. We also found that ORF9b stabilization by USP29 enhanced the virulence of VSV-eGFP and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). Moreover, we observed that the mRNA level of USP29 in SARS-CoV-2 patients was higher than that in healthy people. Our findings provide important evidence indicating that targeting USP29 may effectively combat SARS-CoV-2 infection. IMPORTANCE Coronavirus disease 2019 (COVID-19) is a current global health threat caused by SARS-CoV-2. The innate immune response such as type I IFN (IFN-I) is the first line of host defense against viral infections, whereas SARS-CoV-2 proteins antagonize IFN-I production through distinct mechanisms. Among them, ORF9b inhibits the canonical IκB kinase alpha (IKKɑ)/ß/γ-NF-κB signaling and subsequent IFN production; therefore, discovering the regulation of ORF9b by the host might help develop a novel antiviral strategy. Posttranslational modification of proteins by ubiquitination regulates many biological processes, including viral infections. Here, we report that ORF9b is ubiquitinated and degraded through the proteasome pathway, whereas deubiquitinase USP29 deubiquitinates ORF9b and prevents its degradation, resulting in the enhancement of ORF9b-mediated inhibition of IFN-I and NF-κB activation and the enhancement of virulence of VSV-eGFP and SARS-CoV-2 trVLP.

The Role of Deubiquitinases in Virus Replication and Host Innate Immune Response.

As a critical post-translational modification, ubiquitination is known to affect almost all the cellular processes including immunity, signaling pathways, cell death, cancer development, and viral infection by controlling protein stability. Deubiquitinases (DUBs) cleave ubiquitin from proteins and reverse the process of ubiquitination. Thus, DUBs play an important role in the deubiquitination process and serve as therapeutic targets for various diseases. DUBs are found in eukaryotes, bacteria, and viruses and influence various biological processes. Here, we summarize recent findings on the function of DUBs in modulating viral infection, the mechanism by which viral DUBs regulate host innate immune response, and highlight those DUBs that have recently been discovered as antiviral therapeutic targets.